For tissue samples, free glutathione (the sum of GSH and GSSG levels) was quantified using a GSH and GSSG assay kit (Beyotime, Nantong, China) following the manufacturer’s instructions. The measurements of GSH and GSSG were monitored by a microplate reader (Model 680, Bio-Rad, Hercules, CA, United States). The GSH/GSSH ratio was then calculated. For subcellular compartments, because it’s difficult to preserve glutathione pool against oxidation during the conventional extraction and purification procedures required to obtain subcellular fractions, the absolute concentrations of free glutathione are not possible to be precisely determined (García-Giménez et al., 2013 (link); Noctor et al., 2016 (link)), the subcellular levels of free glutathione per gram organelle protein were measured as ratio of A relative to J by using the thiol-group reaction strategy with a GSH and GSSG assay kit (Beyotime) (Galkina et al., 2017 (link)). The isolation of soluble and cell wall conjugated GGT was described by Destro et al. (2011) (link). The activity of GCL and GGT was determined using a GCL assay kit (Jian Cheng, Nanjing, China) and a GGT assay kit (Solarbio, Beijing, China), respectively. The assay of SAT and CS activity was conducted according to Watanabe et al. (2008) (link) and Nguyen et al. (2012) (link), respectively.
Gsh and gssg assay kit
Manufactured by Beyotime
Sourced in China, Japan
The GSH and GSSG Assay Kit is a laboratory tool designed to measure the levels of glutathione (GSH) and glutathione disulfide (GSSG) in biological samples. It provides a quantitative assessment of these important antioxidants, which play a crucial role in cellular redox homeostasis.
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254 protocols using gsh and gssg assay kit
1
Quantifying Glutathione Metabolism and Enzyme Activities
2
Glutathione Assay in Gastric Cancer Cells
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GSH and GSSG Assay kit (catalog no. S0053; Beyotime Institute of Biotechnology) was used to detect the level of GSH/GSSG in MGC-803 and HGC-27 cells. Following treatment with PITC (0, 50 and 150 µM for MGC-803; 0, 20 and 60 µM for HGC-27) for 12 h at 37°C, the cells were harvested and counted. Cells were transferred to new tubes to ensure equal the cell numbers in each group and washed with PBS. The protein removal solution was used to resuspend cells. The tubes were placed in liquid nitrogen and 37°C water twice for fast freezing and thawing to split the cell membrane and subsequently placed in 4°C for 5 min and centrifuged at 10,000 × g at 4°C for 10 min. The supernatants were collected to detect the amount of total (t)GSH. GSH and GSSG Assay kit (Beyotime Institute of Biotechnology) was used to detect tGSH following the manufacturer's instructions. Briefly, GSH assay working solution and samples were added to 96-well plates and incubated at room temperature for 5 min; reduced nicotinamide adenine dinucleotide phosphate was added into the reactive system and incubated for 25 min at room temperature. Absorbance at 412 nm was measured by a microplate reader.
3
Glutathione Quantification in Cultured Cells
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The cells were cultured in SC-URA medium and collected when the OD600 reached 4.0 and then were washed by PBS. The mixture of cells were added to three times the volume of deproteinization buffer M from the GSH and GSSG Assay Kit (Beyotime S0053, China) and then were alternately subjected to multi-gelation twice in liquid nitrogen and 37 °C water. After centrifugation (12,000 × g, 10 min, 4 °C), the supernatant was collected for GSH and GSSG determination. The levels of GSH and GSSG were determined using a GSH and GSSG Assay Kit (Beyotime S0053) according to the manufacturer’s protocol.
4
Assessing Cell Glutathione Levels
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3 × 105 LLC cells were seeded in a 6‐well plate and treated with different interventions. After 48 hours, the cell suspension was collected and subjected to 3 repeated freeze‐thaw cycles for subsequent experiments. The GSH and GSSG assay kits were used according to the manufacturer's instructions (Beyotime Biotechnology, China) to determine the concentration.
5
Quantification of Intracellular GSH/GSSG
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Intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) contents were determined by GSH and GSSG Assay Kits (Beyotime, S0052). After 10 μl of supernatants were mixed with 50 μl NADPH (0.5 mg/ml) and 150 μl substrate solution, absorbance was read at 412 nm every 5 min for a total of 25 min. Then, GSH and GSSG contents were calculated according to standard curve.
6
Glutathione Assay Protocol
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GSH and GSSG assay kits (Cat. #S0053, Beyotime, Shanghai, China) were used to measure intracellular and tissue GSH concentrations. In brief, cells and fresh tissues were collected, and the experimental data were determined with a microplate reader according to the kit instructions. These experiments were performed in triplicate. The data were expressed as a percentage relative to the control group.
7
Intracellular GSH Quantification in HeLa Cells
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HeLa cells (5.5 × 105) were spread in 6-well plates, and intracellular GSH was detected using the GSH and GSSG assay kits (Beyotime, Biotechnology, China). The cells were digested down with trypsin after treatment with compound 3a . Afterwards, the cells were lysed using the freeze-thaw method, and the lysate was centrifuged for 5 min to collect the supernatant. The absorbance was measured at 412 nm with a microplate reader, and then the GSH content was calculated.
8
Measuring Glutathione Levels in A375 Cells
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Levels of GSH in A375 cells after treatment with Cu6NC were evaluated with GSH and GSSG assay kits (Beyotime Biotechnology, Jiangsu, China). In short, A375 cells were seeded in 6-well dishes (2 × 105 cells per well) and treated with different concentrations of Cu6NC (0, 2, 8 and 16 μM) for 6 h. Then, the cells were collected with centrifugation, and the supernatant was discarded. Samples were added protein remover M (10 mg/30 μL), subjected to 3 cycles of freezing–thawing, and then centrifugated at 1000g for 10 min at 4 °C. The supernatant was reserved for GSH measurement with GSH and GSSG assay kits according to the manufacturer’s protocol.
9
Quantifying Oxidative Stress Markers
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GSH and GSSG Assay Kits (Beyotime, S0053, Shanghai, China) were used to measure the content of GSH, and then they were standardized to tissue weight. With the indicated treatments, the LPO MDA Assay Kit (Beyotime, S0131S, Shanghai, China) was used to determine the MDA contents. Briefly, the samples were centrifuged after being treated with lysis buffer, then supernatants were mixed with TBA detection solution to measure the absorbance at 532 nm, and then the contents of MDA were calculated according to the standard curve. Finally, the data were standardized to tissue weight.
10
Assessing Iron-Mediated Toxicity in Gardnerella vaginalis
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Five to seven colonies of G. vaginalis on Columbia blood agar plates were randomly selected and inoculated into 5 mL of BHIs culture and was cultured at 37 ℃ with 5% CO2 for 12–18 h. One day post‐inoculation, required amounts of G. vaginalis bacterial solution was taken and transferred into fresh medium at a ratio of 1:100 and cultured at 37 ℃ with 5% CO2 for 6–8 h. When the OD600 reached a value of 0.5, 100 µL of bacterial inoculation was mixed with BHIs (900 µL) as the control. An additional 100 µL of bacterial inoculation was mixed with D‐Fe3S4 and BHIs (800 µL) as the experimental group. After incubation at 37 ℃ for certain time, bacterial viability was checked by plating bacteria with proper dilution and calculating the bacterial number in CFU mL–1. The inhibition assays using either FeCl2, Na2S4, EDTA, ferrostatin‐1 or other inhibitors were conducted in the same system and using the same procedure. Glutathione levels were measured using GSH and GSSG assay kits (Beyotime, China). The fluorescent probe Bodipy581/591‐C11 was used to measure the lipid peroxidation level of bacteria. ATP Content Assay Kit and Micro Glucokinase (GLK) Assay Kit (Solarbio, China) were used to measure ATP and glucokinase activity levels.
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